The research program described in this application deals with the mechanism of protein import into the mitochondrion. The experimental model is the budding yeast Saccharomyces cerevisiae, which is an ideal model for mammalian systems because protein import is highly conserved. Previous work has identified a new import pathway for proteins of the mitochondrial inner membrane, which is distinct from the pathway used by precursors with an amino-terminal targeting presequence. Components of this import pathway include the soluble Tim8/Tim13 and Tim9/Tim10 complexes of the intermembrane space and the TIM22 Complex (Tim12, Tim22, and Tim54) of the inner membrane. Mutations in DDP1 (deafness/dystonia protein; homologous to Tim8) cause the human disease Mohr-Tranebjaerg Syndrome, which is most likely caused by a defective protein import machinery. The objective of the research proposed here is to define the molecular mechanisms of this import pathway with a combined biochemical, biophysical and genetic approach. Specifically, the inner membrane substrates and their motifs, which are recognized by the Tim8/Tim13 and Tim9/Tim10 complexes, will be determined. Moreover, the mechanism by which the Tim8/Tim13 and Tim9/Tim10 complexes escort the substrates to the inner membrane will be elucidated. Using temperature-sensitive tim12 and tim22 mutants, additional components of the TIM22 complex will be identified and characterized with respect to location and function. The proposed project will expand fundamental knowledge about the mechanism of protein insertion into the mitochondrial inner membrane, extending present studies that have focused generally on how proteins reach the soluble compartments of the mitochondria. Also, these studies will contribute to the basic understanding of how proteins insert into membranes and how defects in mitochondrial biogenesis can contribute to mitochondrial diseases.